Lead toxicity, defense strategies and associated indicative biomarkers in Talinum triangulare grown hydroponically

Chemosphere 89 (2012) 1056–1065

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Lead toxicity, defense strategies and associated indicative biomarkers in Talinum triangulare grown hydroponically Abhay Kumar a, M.N.V. Prasad a,⇑, Oksana Sytar a,b a b

Department of Plant Sciences, University of Hyderabad, Hyderabad 500 046, India Department of Plant Physiology and Ecology, Kyiv National University of Taras Shevchenko, Kyiv 01033, Volodymyrskya str. 64, Ukraine

h i g h l i g h t s " Talinum triangulare in hydroponics was investigated for Lead (Pb) toxicity and tolerance biomarkers. " Lead exposure resulted in oxidative stress in T. triangulare. " Roots accumulated more Pb than stems, and below detectable level in leaves. " Increased MDA and

a-tocopherol, decreased GSH/GSSG ratio serve as biomarkers of Pb exposure.

" Lead toxicity reduced water transport and nutrient elements.

a r t i c l e

i n f o

Article history: Received 28 January 2012 Received in revised form 16 April 2012 Accepted 16 May 2012 Available online 20 June 2012 Keywords: Ceylon spinach Defense response Energy-dispersive X-ray spectrometry Non-enzymatic antioxidants Oxidative stress Toxicity biomarkers

a b s t r a c t Talinum species have been used to investigate a variety of environmental problems for e.g. determination of metal pollution index and total petroleum hydrocarbons in roadside soils, stabilization and reclamation of heavy metals (HMs) in dump sites, removal of HMs from storm water-runoff and green roof leachates. Species of Talinum are popular leaf vegetables having nutrient antinutrient properties. In this study, Talinum triangulare (Jacq.) Willd (Ceylon spinach) grown hydroponically were exposed to different concentrations of lead (Pb) (0, 0.25, 0.5, 0.75, 1.0 and 1.25 mM) to investigate the biomarkers of toxicity and tolerance mechanisms. Relative water content, cell death, photosynthetic pigments, sulphoquinovosyldiacylglycerol (SQDG), anthocyanins, a-tocopherol, malondialdehyde (MDA), reactive oxygen species (ROS) glutathione (GSH and GSSG) and elemental analysis have been investigated. The results showed that Pb in roots and shoots gradually increased as the function of Pb exposure; however Pb concentration in leaves was below detectable level. Chlorophylls and SQDG contents increased at 0.25 mM of Pb treatment in comparison to control at all treated durations, thereafter decreased. Levels of carotenoid, anthocyanins, a-tocopherol, and lipid peroxidation increased in Pb treated plants compared to control. Water content, cells death and elemental analysis suggested the damage of transport system interfering with nutrient transport causing cell death. The present study also explained that Pb imposed indirect oxidative stress in leaves is characterized by decreases in GSH/GSSG ratio with increased doses of Pb treatment. Lead-induced oxidative stress was alleviated by carotenoids, anthocyanins, a-tocopherol and glutathione suggesting that these defense responses as potential biomarkers for detecting Pb toxicity. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Lead (Pb) ranks second among all the hazardous heavy metals (HMs). Majority of the emissions to the environment are from metallurgy (mining, smelting, metal finishing and combustion of coal), energy production (gasoline, battery and power plants), fertilizers, pesticides, lead-containing paints, explosives, microelectronics, sewage sludge and waste waters (Sharma and Dubey, 2005; Sengar et al., 2008; Bindu et al., 2010). Lead is a non-essential for plant ⇑ Corresponding author. Tel.: +91 40 23011604; fax: +91 40 23010120/145. E-mail address: [email protected] (M.N.V. Prasad). 0045-6535/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2012.05.070

metabolism and exhibit varied degrees of phytotoxicity including adverse effects to photosynthesis (Tang et al., 2009). Lead accumulation in different parts of plant is not only depends upon the exogenous concentration, but also on the phytochemistry and soil physico-chemical conditions (Reddy et al., 2005; Mishra et al., 2006). Excess Pb in plant cells cause molecular damage either directly or indirectly through the formation of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical (OH) and superoxide radicals (O 2 ) (Sharma and Dubey, 2005; Sengar et al., 2008; Wang et al., 2010; Pourraut et al., 2011). The ROS changes membrane structure and function by attacking on the

A. Kumar et al. / Chemosphere 89 (2012) 1056–1065

polyunsaturated fatty acid of membrane lipids, initiating lipid peroxidation and altered cell biochemical activities (Reddy et al., 2005; Sun et al., 2010; Maldonado-Magaña et al., 2011). As a non-redox active metal, Pb can replace essential metals or cofactors at enzyme active site causing imbalance in cellular redox status. Plants exposed to Pb showed changes in photosynthetic rate due to distortion of chloroplast ultrastructure, impairments of chlorophyll synthesis, imbalance in plastoquinone, reduced minerals uptake and obstructed electron transport (Sharma and Dubey, 2005; Sengar et al., 2008; Cenkci et al., 2010; Pourraut et al., 2011) Chlorophylls and total carotenoid are considered as simple and reliable indicators of Pb-induced phytotoxicity in higher plants (Krupa et al., 1996). ROS production in higher plants not only affects the chlorophylls but also changes the lipids such as sulphoquinovosyldiacylglycerol (SQDG) compositions. SQDG is the deacylation product of sulpholipid and comprises up to 24% of the four major chloroplast lipids. In higher plants approximately 40% of the thylakoid SQDG was tightly associated with LHC II (Harwood and Okanenko, 2003). SQDG is implicated in regulation of light energy distribution between the two photosystems via the phosphorylation of the mobile antennae (Larsson and Andersson, 1985). Plant cells are protected against ROS by enzymatic and nonenzymatic antioxidative defense systems. Non-enzymatic antioxidative system, involves carotenoids, phenols, flavonoids and glutathione etc., are known to play an important role in scavenging ROS. Anthocyanins (in vacuole) and a-tocopherol (membrane associated) are major secondary metabolites found in the plants against oxidative stresses (Araceli et al., 2009; Hernández et al., 2009). Their antioxidant activities are due to by capturing free radical ions by donation of phenolic hydrogen atoms and protect the plant cells from adverse effects of abiotic stresses (Hernández et al., 2009). Another important property of anthocyanins is that they react with alkaloids, other flavonoids, organic acids, biomolecules and metals forming complexes and stabilizing the color in plants (Araceli et al., 2009). The most active form of vitamin E is a-tocopherol, which is capable of scavenging free oxygen radicals and lipid peroxides. It has been reported that abiotic stress factors might alter tocopherol levels in plant tissues (Collin et al., 2008; Yusuf et al., 2010). In addition to flavonoids, glutathione is widely distributed in living cells and the oxidized form of glutathione (GSSG) is readily converted in to reduced form (GSH) by the enzyme glutathione reductase (GR) (Wang et al., 2010; Maldonado-Magaña et al., 2011). The tripeptide GSH (c-Glu– Cys–Gly) is playing a key role in scavenging ROS and protecting membrane to damage by these free radicals. In plant cells, metal detoxification mechanisms involve synthesis of the phytochelatins (PCs), and specific metal binding ligands (Gupta et al., 1995, 1999; Mishra et al., 2006; Anjum et al., 2012). Talinum triangulare (Jacq.) Willd (Ceylon spinach, Talinaceae) is a terrestrial succulent herbaceous plant is widely used, as ornamental and green leafy vegetable, in India, Africa, America and Arabia (Nyffeler and Eggli, 2010). It can be propagated through stem cuttings in hydroponic media (Kumar and Prasad, 2010). The extensively produced adventitious roots of plants are having the potential to absorb toxic substances from aqueous media. Plants that accumulate metals from contaminated media are useful for risk assessment studies of toxic metals (Gupta et al., 1995, 1999; Reddy et al., 2005; Tang et al., 2009). T. triangulare are reported to accumulate HMs from dump sites including green leachates and storm water runoff from (Rajkumar et al., 2009; Uwah et al., 2009; Aitkenhead-Peterson et al., 2011). In hydroponic experiments it produces adventitious roots and shoots that are convenient to harvest and for further analysis. Hydroponics are advantageous over soil (in pot culture) since the roots directly exposed to water and the plant metal interactions are direct without

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any interference. Thus, in hydroponics, plant roots exhibit true responses of Pb toxicity. Lead toxicity and tolerance on various biological parameters have been investigated in a number of experimental model systems (Table S1). We investigated Pb-induced toxicity and tolerance biomarkers in T. triangulare in terms of oxidative damage and associated ROS production and elemental analysis by energy-dispersive X-ray spectrometer to provide insight on Pb-toxicity defense strategies and associated indicative biomarkers. 2. Materials and methods 2.1. Plant description T. triangulare is a succulent herb that grows in shallow soil in rocky outcrops. Plants required for these experiments were collected from field gene bank, University of Hyderabad, India. T. triangulare was propagated through stem cuttings (30–50 mm diameter and 16–20 cm height), which gives extensive adventitious roots, leaves and vegetative propagules in hydroponic media under laboratory conditions in about three weeks (Kumar and Prasad, 2010). 2.2. Pb treatment in hydroponic experiment Excised stem cuttings of T. triangulare were placed in conical flask containing 100 mL of modified Hoagland’s media in plant growth chamber at 16/8 h d/night and at 28 ± 2 °C, (Hoagland and Arnon, 1950). Phosphate and sulfate were replaced by chloride and nitrate and the pH of the modified solution was maintained at 4.8 to avoid the precipitation of nutrient media. The nutrient media was replaced every 3 d to provide a fresh dose of nutrient elements. After 3 weeks of acclimatization, uniform stem cuttings with adventitious roots and leaves were selected and treated separately with Pb(NO3)2 at different Pb concentrations of 0 (control), 0.25, 0.5, 0.75, 1.0 and 1.25 mM and maintained under above conditions. Roots, leaves and stems were separated from treated plants after 1, 3, 5 and 7 d were used for the study of various parameters. All the treatments were repeated at least three times. 2.3. Quantification of Pb accumulation in plant Metal treated plants roots were washed thoroughly with 0.1 M HNO3 to remove metals adsorbed to the surface. Roots, stems and leaves were separated and oven dried at 80 °C for 2 d. Dried plant materials were acid digested with 4 mL of HNO3/HClO4 (3:1 v/v) and the digested material was dissolved in the 10 mL of 0.1 N HNO3. This solution was analyzed for Pb content using atomic absorption spectrophotometer (GBC 932 plus, Australia). 2.4. Detection of relative water content (RWC) Plant leaves were freshly weight (FW) and were kept in water at 4 °C for 2 h to know turgid weight (TW). After that leaves were oven dried at 60 °C till completely dry followed by the estimation of dry weight (DW). The relative water content (RWC) was calculated using RWC (%) = [(FW  DW/TW  FW)]  100 (Gao et al., 2010). 2.5. Detection of cell death To determine changes in viability of cells after Pb treatment, 0.1 g of freshly harvested leaves were stained with 0.25% (w/v) aqueous solution of Evans blue for 15 min (Baker and Mock, 1994). After washing with miliQ water for 30 min, leaves were

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excised and soaked with 3 mL of N,N-dimethylformamide for 1 h at room temperature. The absorbance of released Evans blue was measured at 600 and 652 nm and was expressed as A600 after correction for chlorophyll. 2.6. Photosynthetic pigments estimation Extraction and estimation of chlorophyll and carotenoid contents were carried out by the method of Arnon (1949) and Duxbury and Yentsch (1956), respectively. T. triangulare leaves (0.1 g) were ground in 5 mL chilled 80% acetone in dark. After centrifugation at 5000g for 10 min at 4 °C, absorbance of supernatant was taken at 480, 645 and 663 nm.

homogenized in 3 mL 0.1 M tris buffer containing 0.3 M NaCl. After that 2 mL of 20% trichloroacetic acid (TCA) containing 0.5% 2-thiobarbituric acid (TBA) and 2 mL 20% TCA. Mixture was heated at 95 °C for 30 min and then homogenate was centrifuged at 10 000g for 5 min. Absorbance of the supernatant was taken at 532 nm. MDA was calculated by using extinction coefficient of 155 mM1 cm1. Level of ROS, hydrogen peroxide (H2O2), in leaves of T. triangulare treated with 0, 0.25 and 1.25 mM of Pb was determined by 5 lM 20 ,70 -dichlorodihydrofluorescein diacetate (DCFDA) dye with using confocal microscope (Leica TCS SP2 AOBS Microscope, Germany) at 480 nm excitation and 520 nm emission wavelengths.

2.7. Sulphoquinovosyl diacylglycerol estimation

2.11. Glutathione estimation

Plant leaves (0.2 g) were homogenized with 0.5 g glass powder and 0.5 g Na2(SO4). The homogenate was transferred to a glass column and 3 mL acetone was added for filtration. To the filtrate 1 mL hexane: benzene (4:1 v/v) and 2 mL H2O was added and centrifuged at 5000 g for 5 min at room temperature (RT). After centrifugation 1 mL of the bottom layer was added to 1 mL of 0.01% azure (prepared in acetone) and 2 mL benzene, and again centrifuged at 5000g for 5 min at RT. The upper layer was collected and absorbance was measured at 610 nm. The calculation of SQDG was based on the standard curve of sodium dodecylsulfate according to Kean (1968).

Fresh leaf (0.5 g) was ground in 4 mL of 0.1 M sodium phosphate–EDTA buffer (pH 8.0) containing 25% H3PO3. The homogenate was centrifuged at 15 000g for 20 min at 4 °C. In supernatant total glutathione (GSH and GSSG) content was determined fluorometrically after 15 min incubation with o-phthaldialdehyde (OPT) (Hissin and Hiff, 1976). Fluorescence intensity was measured at 420 nm after excitation at 350 nm on a FluoroMax 3 fluorescence spectrophotometer. Total GSH and GSSG in leaves were calculated from the standard curve.

2.8. Anthocyanins estimation Anthocyanins were extracted from 0.5 g of leaves with 10 mL of mixture of n-propanol:HCl:H2O (18:1:81, v/v/v). The samples were heated in boiling water bath for 30 min and then they were incubated for 24 h in the dark at 4 °C. Extracts were filtered and absorbance was taken at 535 and 650 nm. Anthocyanins contents were calculated according to Lange et al. (1971) and Bette and Kutschera (1996) and were expressed as A535 g1 fresh weight after correction for chlorophyll: A535 = A535  0.22  A650. 2.9. a-Tocopherol estimation Concentration of a-tocopherol was measured in leaves as described by Szyman´ska and Kruk (2008) with some modifications. Fresh leaves (1 g) were homogenized in mortar with 20 mL of ice chilled methanol (HPLC grade). The extracts were centrifuged at 10 000g for 15 min at 4 °C. The supernatant were collected and was filtered through 0.2 lm millipore filter and the filtrate was stored in ice in dark. The a-tocopherol content in filtrate was determined by using High Performance Liquid Chromatography (HPLC) with a C18 reverse-phase column (250  4.60 mm, 5 lm Phenomenax, UK) at a flow rate of 1 mL min1, using isocratic solvent system of methanol: ethyl acetate (1:4 v/v) as an eluant. Thirty microliters of the samples were injected by a microliter syringe and a-tocopherol content was quantified by UV–visible detector system at 295 nm (LC-10 AT VP Shimadzu, Japan). Peak identification of analyzed leaf extract was performed by comparing with the retention time of standard a-tocopherol. Chromatogram peak area of leaf sample was integrated and the concentration of a-tocopherol was calculated with regard to the area of the corresponding standard a-tocopherol. 2.10. Lipid peroxidation and ROS estimation Lipid peroxidation in leaves was determined as a function of malondialdehyde (MDA) content following the method of Heath and Packer (1968) with slight modifications. Leaves (0.2 g) were

2.12. Elemental analysis by energy dispersive spectroscopy (EDS) Energy-dispersive X-ray spectroscopy (EDS) is an analytical technique used for the elemental/chemical analysis of specimen. A thin transverse section of leaves treated with 0, 0.25 and 1.25 mM of Pb were prepared. Sectioned tissues were dehydrated, mounted on aluminum stubs, coated with gold-palladium and elemental analyses were done with EDS (Oxford instruments) coupled with field emission scanning electron microscope (FESEM, Ultra 55-Carl Zeiss) and wavelength dispersive X-ray spectroscopy (WDS, Oxford instruments). The EDS analysis was carried out with INCA analyzer software at an operating voltage of 20 KV and working distance of 8.5 lm.

2.13. Statistical analysis The experiment was repeated at least three times. The mean values ± standard errors (SE) are reported in the figures. Significant differences of these data were calculated using analysis of variance (ANOVA–Duncan’s multiple test, SIGMASTAT 9.0). P values 6 0.05 were considered significantly different and denoted by different letters.

3. Results 3.1. Lead accumulation in plant T. triangulare was found to be sensitive to Pb exposure and the Pb accumulation was calculated in tissues on dry weights (dw) basis. When the T. triangulare cuttings were raised under increasing concentration of Pb, the Pb accumulation in plant parts increased significantly as compared to control (Table 1). Roots were the main accumulation site as they absorbed much higher quantities (19.9 mg g1 dw) than stems (2.67 mg g1 dw) while in leaves, Pb accumulation has been observed below detectable limit after 7 d of treatment.

A. Kumar et al. / Chemosphere 89 (2012) 1056–1065 Table 1 Lead accumulation in roots and stem of T. triangulare grown for 7 d in different doses of Pb(NO3)2. Pb concentration (mM)

0 0.25 0.50 0.75 1.0 1.25

Concentration of Pb taken up by plant (mg g1 dw) Roots

Stem

ND 3.14 ± 0.3a 7.85 ± 1.2b 13.5 ± 1.4c 15.5 ± 1.6c 19.9 ± 0.9d

ND 0.81 ± 0.04a 1.18 ± 0.28ab 1.70 ± 0.17b 1.88 ± 0.16c 2.67 ± 0.07d

In a column, means followed by different letter(s) are significantly differ (P 6 0.05) according to DMRT. ND: not detectable.

3.2. Lead-induced changes in relative water content Relative water content in the leaves of T. triangulare, after 7 d of Pb treatment, decreases gradually (Fig. 1). RWC significantly decreased by 6.33% at 0.25 mM to 19.6% at 1.25 mM of Pb, respectively in comparison to control. The water imbibition rates also decreased steadily with increasing Pb concentration. 3.3. Lead-induced cell death Pb-induced oxidative damage in T. triangulare leaves were confirmed by the staining with Evans blue (Fig. 1). A dose-dependent response for cell death was also evident for treatments with different concentrations of Pb. There was no significant difference with control in 0.25 mM Pb treated plant leaf. Evans blue accumulation in leaf was significantly increased maximum by 137% at 1.25 mM Pb treatment in respect to control. 3.4. Lead-induced changes in photosynthetic pigments The chlorophylls concentrations showed an inverted U-shaped curve with the increasing Pb concentrations after 1 d of treatment period. Chlorosis of leaves was observed after 5 d at higher Pb concentrations, which was further increased with subsequent treatment period. In leaves decreased chlorophylls and carotenoid concentrations were observed at higher concentrations (0.75, 1.0 and 1.25 mM) of Pb. The highest concentration of Chl a, b and total chlorophyll were found at 0.5 mM after 1 d and at 0.25 mM after 3, 5 and 7 d of Pb treatment. Contents of Chlorophyll a, b and total chlorophyll were significantly (P 6 0.05) increase by 17%, 37%

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and 24% over control at 0.50 mM of Pb after 1 d, respectively. After 7 d at 1.25 mM of Pb concentration, there were ca. 19%, 8% and 14.8% decrease in Chlorophyll a, b and total chlorophyll in comparison to control, respectively (Fig. 2A). On the other hand, the carotenoid contents were significantly different (Duncan’s multiple test, P 6 0.05) at 0.25, 0.5 and 0.75 mM Pb among all treatment periods in comparison to control. After 1 and 3 d of treatment at 0.5 and 0.25 mM of Pb, carotenoid content was significantly increased up to 26% and 40% in comparison to control, respectively (Fig. 2B).

3.5. Lead-induced changes in SQDG concentration The SQDG concentration significantly (P 6 0.05) increased at 0.25, 0.5 and 0.75 mM and decreased at 1.0 and 1.25 mM of Pb treatment in comparison with control after 1 d (Fig. 3). The tendencies to decrease the SQDG concentrations were observed at 1.0 and 1.25 mM of Pb after all treatment periods. However, after 5 and 7 d the SQDG concentration showed dose-dependent reduction at higher concentration of Pb. After 1 d at 1.0 and 1.25 mM of Pb, SQDG concentrations were decreased and at control level, but in the subsequent period increase in SQDG concentrations have been observed. After 7 d SQDG concentrations at 0.75, 1.0 and 1.25 mM of Pb significantly (P 6 0.05) decreased to ca. 119%, 226% and 212% in comparison to control, respectively, but with 0.5 mM of Pb concentration decreased to ca. 85%, 176% and 164%, respectively (Fig. 3).

3.6. Lead-induced changes in anthocyanins concentration In our experiment, anthocyanins accumulation was higher at 0.25 mM of Pb compared with control after all treatment periods (Fig. 3). In comparison to control 1.7 fold reductions in anthocyanins have been observed at 1.25 mM of Pb treatment after 1 d. But interestingly after 7 d of Pb exposure, anthocyanins were increased and almost at the control level. Significant (at 0.25 mM of Pb) and insignificant (at 0.5–1.25 mM of Pb) increases in anthocyanins were observed after 3 d of treatment periods. The concentrations of anthocyanins were (significant, P 6 0.05) increased at 0.5 and 0.75 mM of Pb treatment after 5 and 7 d of treatment period. Lead induced an intense anthocyanins accumulation, especially after the seventh day of treatment. The maximum concentrations of anthocyanins were observed at 0.5 and 0.75 lM of Pb and were 1.7 and 1.6 fold increased than control, respectively (Fig. 3).

Fig. 1. Relative water content and cell death measurement in T. triangulare leaf after 7 d of lead treatment. All the values are mean of triplicates ± SE. ANNOVA significant at P 6 0.001. Different letters indicate significantly different values (P 6 0.05).

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Fig. 2. Concentrations of chlorophyll a, chlorophyll b and total chlorophyll (A) and carotenoids (B) in T. triangulare after treated with lead. All the values are mean of triplicates ± SE. ANNOVA significant at P 6 0.001. Different letters indicate significantly different values (P 6 0.05).

Fig. 3. Sulphoquinovosyl diacylglycerol (SQDG) concentration and accumulation of anthocyanins in leaves of T. triangulare treated with lead. All the values are mean of triplicates ± SE. ANNOVA significant at P 6 0.001. Different letters indicate significantly different values (P 6 0.05).

3.7. Lead-induced changes in a-tocopherol concentration The HPLC analysis of leaf extracts were identified by comparison of retention time and UV absorption spectra with those obtained for corresponding standards. Peaks were observed at 3.42 ± 0.025 min for a-tocopherol (Fig. 4 inset). Results showed

that the a-tocopherol content gradually increased in treated plants compared to control. But after 7 d significant (P 6 0.05) increase in the a-tocopherol level has been observed at higher (0.75– 1.25 mM) doses of Pb. The levels of increased a-tocopherol ranges between 1.2 and 2.2 fold at 0.25–1.25 mM of Pb in comparison to control (Fig. 4).

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3.8. Lead-induced lipid peroxidation and ROS production

3.9. Lead-induced changes in glutathione

Experimental results of MDA estimation, an indicator of lipid peroxidation, showed that the MDA concentrations were significantly (P 6 0.05) increased than control after Pb treatment and were directly related to the treatment concentrations. MDA concentration in leaves increased after 7 d due to Pb toxicity and the magnitude of elevation ranged from 1.4 to 2.1 folds at 0.25– 1.25 mM of Pb compared to respective controls (Fig. 4). Seven days of Pb treatment prompted a greater increase in the level of MDA at 1.25 mM and was 1.5 fold increased compared to 0.25 mM of Pb exposure. Pb-induced production of ROS was quantified by DCFDA staining (Fig. S1). A dose-dependent response for ROS production was observed and the level of ROS was higher in plant treated with 1.25 mM of Pb than the plant treated with 0.25 mM of Pb.

The level of reduced (GSH) and oxidized (GSSG) glutathione directly point towards the induction of oxidative stress by Pb (Fig. 5). GSH content in plant leaves were found to increase at all Pb treatment except that 1.25 mM of Pb. The level of GSH was not significantly affected at 0.25 and 1.25 mM of Pb. GSH level showed a maximum value at 0.50 mM of Pb which was about 72% higher than control. GSH content depleted with maximum of ca. 12.5% in plant treated with 1.25 mM of Pb (Fig. 5). On the other hand, GSSG level was found to gradually increased at concentration in comparison to control. Maximum GSSG content was found at 1.25 mM which was ca. 172% higher than control. However, the ratio of GSH to GSSG significantly decline and was inversely related to the Pb treatments.

Fig. 4. a-Tocopherol and malondialdehyde (MDA), as a measure of lipid peroxidation, concentrations in leaves of T. triangulare treated with lead. All the values are mean of triplicates ± SE. ANNOVA significant at P 6 0.001. Different letters indicate significantly different values (P 6 0.05). In inset, chromatograms of a-tocopherol: (A) treated leaf extract (B) standard solution.

Fig. 5. Levels of reduced (GSH) and oxidized (GSSG) glutathione in the leaves of T. triangulare treated with lead. All the values are mean of triplicates ± SE. ANNOVA significant at P 6 0.001. Different letters indicate significantly different values (P 6 0.05).

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3.10. Elemental analysis by EDS

4. Discussion

Results of energy dispersion spectroscopy give the atomic or chemical characteristics of analyzed tissue. As a first barrier to metals toxicity, most plants accumulate metals and nutrients in the roots and restrict its transport to the shoots and leaves. Microanalysis of elements was performed at the same site in 0, 0.25 and 1.25 mM of Pb-treated plant samples. In all sample the Pb concentration was below the EDS detectable limit (Fig. 6). The other elements such as oxygen (O), potassium (K), magnesium (Mg), chlorine (Cl), sodium (Na), silicon (Si) and copper (Cu) were also detected in leaf tissues (Fig. 6). In analyzed sample, O and K were contributed as major elements. Trace elements include Mg, Cl, Na, Si, Cu and Ca. X-ray microanalysis of untreated samples showed high spectral peak for all elements, except for O, in comparison to treated (at both 0.25 and 1.25 mM of Pb) plant. Only calcium (Ca) was observed in leaf sample treated with 1.25 mM of Pb (Table S2). Qualitative percentage composition analysis revealed that the percentages of all elements were decreased in both treated plant leaves except O which was increased in Pb treated plant leaves.

In the present study, the Pb accumulation in the tissues increased as the exogenous Pb concentration increased. The efficiency of metal uptake by plants depends on several processes for metal uptake by roots and its translocation from root to the shoot. Roots absorbed high amount of Pb than stem (Table 1). Probst et al. (2009) also reported that Vicia faba roots absorbed more metals than stems and leaves. The order of metal accumulation was root > leaf > stem for all HMs except for Pb and Cd where the order was root > stem > leaf for Vicia faba (Probst et al., 2009). This order in different species may vary. In our experimental system, no Pb was detected in the leaves of treated plants. Lower intensity of Pb in the stems (Table 1) and below detectable limit in leaves of Pb treated plants confirmed the decreased translocation of Pb within the plants. It is confirmed by Tian et al. (2011) that Pb is rather immobile within the plants, and accumulated mostly in the vascular bundles of the plant tissues and it could not re-translocated in the tissues even after treatment with a metal chelating agent i.e. EDTA (ethylenediaminetetraacetic acid). The immobilization of Pb+2 on T. triangulare leaves could represent a new and attractive characteristic for the development of phytoremediation strategies requiring that the large amount of the toxic substances remain tightly bound to plant tissues. In our experimental system Pb bioaccumulation in roots and stems through Hoagland’s solution represents the Pb accumulation capacities of T. triangulare that are almost equivalent to those of efficient root accumulators of HMs like Lathyrus sativus (Brunet et al., 2008), and Brassicae juncea (Meyres et al., 2008) or less than Sedum alfredii (Tian et al., 2011). It is found that hydroponically cultivated adventitious roots of several terrestrial plants were used for rhizofiltration, a process used for absorbing or precipitating toxic metals effectively from a polluted site (Dushenkov et al., 1995). Adventitious roots of Talinum plants have the potential to absorb HMs and might be useful for a new rhizofiltration system. Relative water content in HM stressed plants is an indicator of phytotoxicity. HMs generally causing a loss of leaf water potential than in non-treated plants (Prasad, 1997; Gao et al., 2010). The results showed that Pb caused phytotoxicity to the plants, which was evidenced by the significant decrease of water content in treated leaves. In the present experiment, RWC decreased with increasing Pb concentrations, and this could be due to decreased water uptake and transport from root to leaf. The damage of membrane integrity was detected by uptake of a non-permeable dye (Evans blue) into the leaf cells, which has been used as an indicator of cell death. Evans blue staining showed that Pb is able to cause death of plant leaf cells. This could be possible due to decline in water content and decrease in nutrient elements contents in leaf of treated plants in comparison to control. Lead exerts adverse effects on the morphology, growth and photosynthetic processes of plants (Sharma and Dubey, 2005; Sengar et al., 2008; Cenkci et al., 2010; Pourraut et al., 2011). In our experiments we observed that the lower concentrations of Pb (0.25 and 0.5 mM) significantly increasing the chlorophylls content over the control at all duration (Fig. 2A). Exposure of low concentration of Pb has an opposite effects on high concentration exposure. In some cases, lower concentrations stimulate metabolic processes and enzymes involved i.e. photosynthesis and enzymatic activities and long term exposure reduced leaf growth, decreased photosynthetic pigments (Parys et al., 1998). Inhibition of the photosynthetic pigment biosynthesis is one of the primary events in plants during HMs stress and decreases in photosynthetic pigment content have also been reported in many plants under Pb stress (Shakya et al., 2008; Cenkci et al., 2010). It was suggested that HMs could interfere with chlorophylls biosynthesis either through the direct

Fig. 6. Energy dispersive X-ray spectral line profile of a transverse section of T. triangulare leaf, control (A), 0.25 mM Pb (B) and 1.25 mM Pb (C). Percent weight elemental composition of the imaged area for the Talinum leaf sample using SEM/ EDS.

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inhibition of enzymatic steps or through the substitution of the central Mg ion (Sengar et al., 2008; Cenkci et al., 2010; Pourraut et al., 2011). Carotenoid serves as antioxidant against free radicals and photochemical damage (Sengar et al., 2008). Thus less effect on carotenoid might represent its supportive role against oxidative stress after 7 d (Fig. 2B). It is possible that at higher Pb concentrations affected the plant water status, causing water deficit, and finally reduced the chlorophylls content. The SQDG defined the chlorophylls orientation in membrane and the changes in sulpholipid (SQDG) quantity under HMs stress as a marker of oxidative stress is known phenomenon and studies on stress responses have shown quantitative and qualitative changes in SQDG (Harwood and Okanenko, 2003). In our experiment, higher (0.75–1.25 mM) Pb concentration causes a decrease in SQDG concentration (Fig. 3). Similar results with decrease of SQDG concentrations were observed with influence of various concentrations of Pb on wheat seedling, leaves and roots which grew in hydroponic cultures (Okanenko et al., 2003). It has also been reported that Cd concentration induced a decrease in SQDG concentration in Brassica napus leaves (Youssef et al., 1998). The most HMs tested cause a decrease of SQDG concentration (Youssef et al., 1998; Pádua et al., 2003). It is possible that under stress conditions competitive use of sulfur for synthesis of sulfur containing cys-rich peptides. It is suppose that the influence of HMs can utilize sulfur preferentially for the synthesis of essential metabolites, such as glutathione and metal binding proteins (phytochelatins and metallothioneins), rather than for SQDG synthesis. Results suggested that in plants, the synthesis of anthocyanins makes it an effective strategy against ROS generation due to Pb stress, but interestingly lower concentrations of Pb (0.25– 0.75 mM) stimulated synthesis of anthocyanins more than higher concentrations (1.0 and 1.25 mM) of Pb (Fig. 3). This indicates that there is a strong correlation between the level of anthocyanins and the presence of HMs in the environment, which further corroborates the results obtained in Zea mays demonstrating that accumulation of anthocyanins increased under HMs stress (Krupa et al., 1996). Anthocyanins not only scavenge free radicals, but they also have the ability to bind HMs and are biosynthesized through the phenylpropanoid pathways, the first enzymatic step of the conversion of phenylalanine to trans-cinnamic acid that catalyzed by phenylalanine ammonium lyase (PAL) (Kitamura et al., 2002). It is suggested that PAL, a key enzyme in the flavonoids synthesis, to be the target of HMs inhibitory influence and finally inhibition of anthocyanins synthesis (Dube et al., 1993). However, it seems to be possible that 0.5 and 0.75 mM of Pb concentration stimulate PAL activity resulting in increased production of anthocyanins which may further bind with metals or metal inducing ions in vacuole and detoxify them from cell. Higher concentration of Pb i.e. 1.0 and 1.25 mM are is toxic and lethal to the plant cell, leading to an inhibition of PAL and thus decrease of the biosynthesis of anthocyanin. The initial increase followed by a decline of PAL activity was also observed under different concentration of Cd in Azolla imbricate (Dai et al., 2006). HPLC data showed that Pb treatment to T. triangulare plant resulted in the enhancement of a-tocopherol level in leaves (Fig. 4). Probably, the gradual increase in a-tocopherol concentration can be associated with the increased oxidative stress, which supports it as a potential biomarker against oxidative stress. Results showed that tocopherol might be involved in the adaptation of plants against Pb stress. The important function of tocopherol is that to scavenging free radicals and protects polyunsaturated fatty acids (PUFAs) and inhibiting the lipid peroxides in membranes. Abiotic stress factors such as HMs have been found to initiate lipid peroxidation and start a chain reaction which further induces the tocopherols biosynthesis. Previous experimental results also showed that increase in tocopherol content in Arabidopsis

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thaliana exposed to Cd and Cu treatment (Collin et al., 2008) and Brassica juncea exposed to Cd (Yusuf et al., 2010), which confers with induced tolerance to plants against HMs stress. After 7 d at higher concentration of Pb, an increase in a-tocopherol concentrations was found in proportion with decrease in chlorophylls content. Previous report suggested that may be the partial degradation of chlorophyll induced the tocopherol content because chlorophyll derived phytol is acting as substrate for tocopherol biosynthesis (Rise et al., 1989). In parallel with increase in a-tocopherol content, lipid peroxidation was also stimulated by Pb stress (Fig. 4). Lipid peroxidation is a biochemical marker of the Pb-induced free radical mediated oxidative damage in Talinum plants. Pb ions are known to induce lipid peroxidation, increasing unsaturated fatty acids and decreasing saturated fatty acids in plants (Mishra et al., 2006; Wang et al., 2010; Maldonado-Magaña et al., 2011; Pourraut et al., 2011). However, the percent increase in MDA content was relatively less (Fig. 4). Generally, free radical generation and membrane damage would be low in tolerant plants and thereby formation of lower levels of MDA content. Therefore, relatively lower degree of increase in MDA content in Talinum due to Pb stress may support its tolerant nature. Lead like other HMs induce oxidative stress conditions in plant cells, which is reflected by an increased production of ROS (Fig. S1). Results showed that in the leaves of Talinum exposed with Pb, there was an increased production of hydrogen peroxide (H2O2) has been observed after 7 d of treatment. It suggested that increased production of ROS is due to the disturbance of electron transport chain in membrane (Sharma and Dubey, 2005; Pourraut et al., 2011). Glutathione (GSH) is usually the most abundant intracellular non-protein thiols. The result of GSH and GSSG estimation in Pb treated plants indicate an alteration in GSH content, and simultaneous increase in GSSG showed a true indication of oxidative stress. Glutathione is one of the major cellular safeguards, maintains the cellular redox status, which plays a protective role as a key antioxidant and a chelating bioligand responsible for detoxification of HMs (Gupta et al., 1995; Mishra et al., 2006; MaldonadoMagaña et al., 2011; Pourraut et al., 2011). In this study a significant increase in GSH content has been observed in leaves of T. triangulare followed by depletion at higher concentration (Fig. 5). At lower concentration induction of GSH accumulation and maintenance of high GSH/GSSG ratio might be attributed to Pb-induced GSH synthesis (Piechalak et al., 2002). Increase in GSSG and decline in GSH show correlation. Ratio of GSH/GSSG decline when Pb concentrations increased, this is evidently due to induction in GSH biosynthesis and rapid reduction of GSSG by the enzyme GR at lower concentration of Pb (Maldonado-Magaña et al., 2011). GSH, as a substrate, synthesize PC due to a reaction catalyzed by phytochelatin synthase (PCS) an enzyme activated by HMs ions including Pb. Decline in GSH at higher concentrations might be attributed to high consumption rate of GSH as antioxidant to combat oxidative stress or for the synthesis of PC. Pb-induced changes in GSH have been reported in Acacia farnesiana (Maldonado-Magaña et al., 2011), Ceratophyllum demersum L. (Mishra et al., 2006), Vicia faba, Phaseolus vulgaris (Piechalak et al., 2002), Hydrilla verticillata and Vallisneria spiralis L. (Gupta et al., 1995, 1999). During the depletion of GSH content, under high Pb stress, formation of Pb–GSH complexes and Pb-induced PC synthesis reduces free Pb concentration in cytoplasm and contributes to suppress the activation of the stress-related responses in plant metabolism (Mishra et al., 2006). The X-ray microanalysis can qualitatively analyze and quantify the total amounts of elements present in tissues. Elemental microanalysis is becoming an important way to understand individual component within tissues deposits, which may be difficult to analyze by other methodology (Nagata, 2004; Shillito et al., 2009). Results of our EDS study showed the notable reduction of elements in

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Pb treated plants in comparison to control (Table S2). From the results, it was shown that X-ray microanalysis using semi-thin sections observed by intermediate high voltage SEM at 20 KV was very useful resulting for quantifying some trace elements in biological specimens. The large decreases observed in Na and Mg concentrations suggest a barrier in transport through the stele or may be due to the damage of the transport system in the plant. Our result corroborate that the observed changes in other biological parameters are due to metal stress or due to decrease of nutrients. 5. Conclusions It is concluded that T. triangulare plants grown hydroponically and exposed to Pb showed that the roots were the main accumulating site as they accumulated more Pb than stem and were below detectable level in leaves. This confirmed that Pb immobilization or decreased translocation within the plants, which represent an attractive aspect for the development of defense strategies. It can be suggest that Pb accumulation potential of T. triangulare were almost equivalent to efficient accumulators. The decrease in RWC, due to decreased water transport to leaf, elements atomic % and increased cell death showed that analyzed Pb concentrations have potential to cause oxidative damage in leaf through increase production of lipid peroxides. Photosynthetic pigments and SQDG contents have been increased at 0.25 and 0.5 mM Pb at early treatment period, which decreased at further concentration and treatment durations. Furthermore, increase level of anthocyanins, duration dependent, and a-tocopherol showed their involvement in the adaptation mechanisms in Pb treated plants. Alteration in glutathione levels showed that GSH maintained an important antioxidant system for the plant to accumulate higher concentration of toxic Pb and survive for longer period. These findings confirm the key role of the toxicity and tolerance strategy of T. triangulare and analyzed defense parameters suggesting that these might be the potential biomarkers for detecting Pb toxicity. Further research on the molecular mechanism(s) of Pb toxicity and its microlocalisation and detoxification mechanism is required to improve our understandings. Acknowledgements A.K. gratefully acknowledged to University of Hyderabad Research Scholarship through the University Grant Commission, New Delhi. O.S. gratefully acknowledges the 3 months research training scholarship in the laboratory of MNVP award by the Centre for International Co-operation in Science (CICS) (Formerly CCSTDS) Chennai, India. Thanks are due to Prof. V.S. Raju, (Emeritus), Kakatiya University, Warangal, India for helpful suggestions on nomenclature of this plant. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2012.05.070. References Aitkenhead-Peterson, J.A., Dvorak, B.D., Volder, A., Stanley, N.C., 2011. Chemistry of growth medium and leachate from green roof system in South-central Texas. Urban Ecosyst. 14, 17–33. Anjum, N.A., Ahmad, I., Mohmood, I., Pacheco, M., Duarte, A.C., Pereira, E., Umar, S., Ahmad, A., Khan, N.A., Iqbal, M., Prasad, M.N.V., 2012. Modulation of glutathione and its related enzymes in plants’ responses to toxic metals and metalloids – a review. Environ. Exp. Bot. 75, 307–324. Araceli, C.-O., Ma de Lourdes, P.-H., Ma Elena, P.-H., Rodríguez, J.A., Carlos Andrés, G.-V., 2009. Chemical studies of anthocyanins: review. Food Chem. 13, 859–871.

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